Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecAmediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and D-galactose (D-Gal). Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl D-galactosamine (D-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-D-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of D-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Cyclic peptide production using a macrocyclase with enhanced substrate promiscuity and relaxed recognition determinants

This project was supported by grants from the ERC (no. 339367, MJ), BBSRC IBCatalyst (no. BB/M028526/1, MJ, WEH), BBSRC FoF (no. BB/M013669/1, MJ, WEH), IBioIC Exemplar (no. 2014-2-4, MJ, WEH), an AstraZeneca studentship (MJ, WEH, LT, KR), the Academy of Finland (no. 259505, DPF) and the SULSA leaders award (WEH). The authors like to thank the Aberdeen Proteomics Facility and the Aberdeen School of Natural and Computing Sciences MS Facility for LCMS analysis. Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S60 and Tables S1–S3. See DOI: 10.1039/c7cc05913bPeer reviewedPublisher PD

Targeting undruggable carbohydrate recognition sites through focused fragment library design

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DCSIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+- dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns

Developing novel glycomimetics targeted to bacterial lectins LecA, LecB and BambL

This dissertation describes the study of new, (non)-carbohydrate based lectin inhibitors as basis for the development of glycomimetics. The ESKAPE pathogens Pseudomonas aeruginosa and Burkholderia ambifaria are both Gram-negative bacteria that are especially dangerous for cystic fibrosis patients and are prominently involved in the patients’ deaths. These bacteria express lectins, which are carbohydrate binding proteins, that are partially involved in adherence, biofilm formation or serve as a virulence factor. P. aeruginosa expresses two calcium-ion dependent lectins, LecA and LecB, whereas BambL, a non-metalated lectin, belongs to B. ambifaria. In this work, various strategies were used for addressing these lectins with newly developed ligands for inhibiting their functions. The development started by either virtual screening or from a ligand of a known co-crystal structure of the desired lectin. The new ligand-hits were then further studied and validated in different biophysical assays leading to new, improved (non)-carbohydrate based lectin inhibitors.This dissertation describes the study of new, (non)-carbohydrate based lectin inhibitors as basis for the development of glycomimetics. The ESKAPE pathogens Pseudomonas aeruginosa and Burkholderia ambifaria are both Gram-negative bacteria that are especially dangerous for cystic fibrosis patients and are prominently involved in the patients’ deaths. These bacteria express lectins, which are carbohydrate binding proteins, that are partially involved in adherence, biofilm formation or serve as a virulence factor. P. aeruginosa expresses two calcium-ion dependent lectins, LecA and LecB, whereas BambL, a non-metalated lectin, belongs to B. ambifaria. In this work, various strategies were used for addressing these lectins with newly developed ligands for inhibiting their functions. The development started by either virtual screening or from a ligand of a known co-crystal structure of the desired lectin. The new ligand-hits were then further studied and validated in different biophysical assays leading to new, improved (non)-carbohydrate based lectin inhibitors.Diese Dissertation beschreibt die Entwicklung neuer, (nicht)-kohlenhydratbasierter Lektininhibitoren als Grundlage für Glycomimetika. ESKAPE-Erreger Pseudomonas aeruginosa und Burkholderia ambifaria gehören beide zu den gramnegativen Bakterien, die in Mukoviszidose-Patienten gravierende Einflüsse haben, die sogar zu deren Tod führen können. Beide Bakterienarten exprimieren verschiedene kohlenhydratbindende Proteine, die so genannten Lektine. Diese sind zum Teil bei der Adhärenz und der Biofilmbildung involviert oder dienen als Virulenzfaktor. P. aeruginosa exprimiert zwei Calciumionen-abhängige Lektine, LecA und LecB, während B. ambifaria ein metallfreies Lektin, BambL, exprimiert. In dieser Arbeit wurden verschiedene Strategien verwendet, um diese Lektine mit neuen Liganden in ihrer Funktion zu inhibieren, dabei wurde entweder vom virtuellen Screening oder von einem Liganden, einer bekannten Co-Kristallstruktur des gewünschten Lektins, ausgegangen. Die neuen Liganden-Hits wurden anschließend in verschiedenen biophysikalischen Assays studiert und validiert. Dies führte zu neuen, zum Teil deutlich verbesserten (nicht)-kohlenhydratbasierten Lektininhibitoren.Diese Dissertation beschreibt die Entwicklung neuer, (nicht)-kohlenhydratbasierter Lektininhibitoren als Grundlage für Glycomimetika. ESKAPE-Erreger Pseudomonas aeruginosa und Burkholderia ambifaria gehören beide zu den gramnegativen Bakterien, die in Mukoviszidose-Patienten gravierende Einflüsse haben, die sogar zu deren Tod führen können. Beide Bakterienarten exprimieren verschiedene kohlenhydratbindende Proteine, die so genannten Lektine. Diese sind zum Teil bei der Adhärenz und der Biofilmbildung involviert oder dienen als Virulenzfaktor. P. aeruginosa exprimiert zwei Calciumionen-abhängige Lektine, LecA und LecB, während B. ambifaria ein metallfreies Lektin, BambL, exprimiert. In dieser Arbeit wurden verschiedene Strategien verwendet, um diese Lektine mit neuen Liganden in ihrer Funktion zu inhibieren, dabei wurde entweder vom virtuellen Screening oder von einem Liganden, einer bekannten Co-Kristallstruktur des gewünschten Lektins, ausgegangen. Die neuen Liganden-Hits wurden anschließend in verschiedenen biophysikalischen Assays studiert und validiert. Dies führte zu neuen, zum Teil deutlich verbesserten (nicht)-kohlenhydratbasierten Lektininhibitoren

Lectin antagonists in infection, immunity, and inflammation.

Lectins are proteins found in all domains of life with a plethora of biological functions, especially in the infection process, immune response, and inflammation. Targeting these carbohydrate-binding proteins is challenged by the fact that usually low affinity interactions between lectin and glycoconjugate are observed. Nature often circumvents this process through multivalent display of ligand and lectin. Consequently, the vast majority of synthetic antagonists are multivalently displayed native carbohydrates. At the cost of disadvantageous pharmacokinetic properties and possibly a reduced selectivity for the target lectin, the molecules usually possess very high affinities to the respective lectin through ligand epitope avidity. Recent developments include the advent of glycomimetic or allosteric small molecule inhibitors for this important protein class and their use in chemical biology and drug research. This evolution has culminated in the transition of the small molecule GMI-1070 into clinical phase III. In this opinion article, an overview of the most important developments of lectin antagonists in the last two decades with a focus on the last five years is give

Lectin antagonists in infection, immunity, and inflammation.

Lectins are proteins found in all domains of life with a plethora of biological functions, especially in the infection process, immune response, and inflammation. Targeting these carbohydrate-binding proteins is challenged by the fact that usually low affinity interactions between lectin and glycoconjugate are observed. Nature often circumvents this process through multivalent display of ligand and lectin. Consequently, the vast majority of synthetic antagonists are multivalently displayed native carbohydrates. At the cost of disadvantageous pharmacokinetic properties and possibly a reduced selectivity for the target lectin, the molecules usually possess very high affinities to the respective lectin through ligand epitope avidity. Recent developments include the advent of glycomimetic or allosteric small molecule inhibitors for this important protein class and their use in chemical biology and drug research. This evolution has culminated in the transition of the small molecule GMI-1070 into clinical phase III. In this opinion article, an overview of the most important developments of lectin antagonists in the last two decades with a focus on the last five years is give

Discovery of N -β- l -Fucosyl Amides as High-Affinity Ligands for the Pseudomonas aeruginosa Lectin LecB

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Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA